Identification of a human VPF/VEGF 3' untranslated region mediating hypoxia-induced mRNA stability.
Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3' untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3' untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3' untranslated region and distinct mRNA-binding proteins in human tumor cells.
Claffey
K P
KP
Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Shih
S C
SC
Mullen
A
A
Dziennis
S
S
Cusick
J L
JL
Abrams
K R
KR
Lee
S W
SW
Detmar
M
M
D001483:Base Sequence; D015687:Cell Hypoxia / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016228:Endothelial Growth Factors / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006207:Half-Life; D006801:Humans; D008222:Lymphokines / Q000235:genetics*; D008545:Melanoma; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D018076:DNA, Complementary; D016228:Endothelial Growth Factors; D008222:Lymphokines; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C039919:RNA, recombinant; D016601:RNA-Binding Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D012313:RNA